Acute chest syndrome (ACS) is a leading cause of morbidity among Sickle Cell Disease (SCD) patients. Therapies that can prevent ACS are highly warranted but the development of such treatments has been stalled by the lack of knowledge of the events that initiate ACS. Both infection and vaso-occlusion are believed to cause ACS; however, the cellular and molecular mechanism of ACS is not completely understood. We propose to use an integrative physiologic approach to test the hypothesis that ACS is caused by occlusion of the pulmonary arterioles by aggregates of neutrophils and platelets and this process of aggregation requires P-selectin and GPIba expressed on membrane extensions known as slings on platelets binding to P-selectin- glycoprotein-ligand-1 (PSGL)-1 and Mac-1, respectively on neutrophil slings. In Aim 1, we will use in vivo multi- photon excitation (MPE) imaging of the lung vasculature in live transgenic SCD mice to prove that intravascular (IV) administration of bacterial lipopolysaccharide (LPS) results in sequential steps of neutrophil accumulation in the pulmonary arteriole, platelet nucleation on accumulated neutrophils, formation of micro-thrombus, abrogation of blood flow and lung injury. The PI has shown previously that neutrophils form slings, which are long membrane extensions decorated with PSGL-1 and CD18. In Aim 2, we will flow blood drawn from SCD patients and race-matched control subjects through in vitro microfluidic channels cultured with human lung endothelial cells and use fluorescence microscopy to prove that platelet nucleation on neutrophils can be prevented by either blocking P-selectin on platelets or Mac-1 on neutrophils or preventing sling formation by neutrophils and platelets. In the Aim 3, we will show that chimeric SCD mice deficient in platelet P-selectin and neutrophil Mac-1 do not develop pulmonary arteriole occlusion and are protected from ACS following IV challenge with LPS. The successful completion of these Aims will demonstrate that ACS can be prevented by inhibiting platelet P-selectin and neutrophil Mac-1.